1. Field of the Invention
The present invention relates to a method and new image acquisition technology for in-gel detection and quantification of proteins. This new platform, called Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis (CUTEDGE™), utilizes differences in fluorescent lifetimes to differentiate between fluorescence from specific protein labels and non-specific background fluorescence, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology.
2. Background of the Invention
“Proteomics” refers to the study of the protein complement of the genome (proteome), a term coined by Marc Wilkins in 1994. Over the past decade, many methodologies for simultaneous quantification of thousands of proteins in a cell or tissue have been developed and utilized for e.g. biomarker discovery or mechanistic studies of cellular processes. Two-dimensional gel electrophoresis (2-DGE) was the first method to be adapted for global proteomics analysis, and still constitutes one of the workhorses in proteomics research. The 2-DGE method involves separation of complex protein samples according to charge in the first dimension and according to size in the second dimension, resulting in a 2-D map of protein spots where ideally each spot corresponds to a single protein species. The protein spots are then visualized with protein stains that bind stoichiometrically to the proteins, thus providing a third dimension that corresponds to protein abundance, which facilitates quantitative proteome analysis.
Problems with large gel-to-gel variations associated with the original 2-DGE technique have been addressed through the incorporation of an internal standard, such as the Differential Gel Electrophoresis (DIGE) and Alexa-Labelled Internal Standard (ALIS) techniques. Both concepts are based on sample proteins and internal standard proteins being labeled with spectrally separated fluorochromes, and co-separated on the same 2-DGE gel. By ratiometric normalization the inter-gel variations can be corrected for, thus greatly improving the quantitative aspects and overall statistical power of the 2-DGE technique.
The major constraint remaining in current 2-DGE methodology is limitations in the sensitivity of detection. The fact that protein abundances in biological samples may span over as much as twelve orders of magnitude puts high demands both on sensitivity and dynamic range of protein stains used in quantitative 2-DGE. Towards this end, fluorescent stains with dynamic ranges of 3-4 orders of magnitude have replaced the use of classical calorimetric staining methods, such as silver and commassie stains with dynamic ranges typically limited to 1-2 orders of magnitude (FIG. 1).
Fluorescent dyes are available both for covalent labeling prior to 2-DGE separation (e.g. CyDyes™, Alexa-dyes), as well as for non-covalent, post-electrophoretic staining procedures (e.g. SYPRO Ruby™, Deep Purple™). However, even the best performing fluorescent probes for protein visualization currently on the market only cover a very small portion of the potential physiological range since physiological protein abundances range from a few molecules up to micromolar concentrations, while detection limits for the state-of-the-art method of minimal DIGE typically are limited to nanograms of protein (FIG. 1).
In current usage of fluorescence for protein detection and quantification in 2-DGE, the excitation of the fluor and the measurement of the resulting emission occur simultaneously. Being time efficient and practical from a technical standpoint, this approach is utilized in both fluorescent scanners and CCD-camera based 2-DGE image acquisition instruments. However, direct fluorescent measurements do not utilize the full potential of these fluorochromes. Biological specimens contain numerous auto-fluorescent components, and the polyacrylamide matrix itself emits background fluorescence to some extent. To optimize the signal-to-noise ratio, it is thus essential to decrease disturbances from background- and autofluorescence.
In current 2-DGE technology, attempts to remove the resulting background are made mathematically through software algorithms used in the post-electrophoretic computer-assisted quantitative analysis. However, we have previously shown that the majority of these background subtraction and correction algorithms alter the data and introduce additional variance into the quantification of protein spot volumes, as well as contribute to a skewed, non-normal distribution (1-4).
Through time-resolved fluorescence (TRF), the origin of a photon can be derived through separation of the decay curves of the various fluorescent species present in a given pixel. TRF is currently used in a number of applications in related fields, primarily microscopy applications to visualize localization, folding dynamics, or movement of proteins in solution (5, 6). Several of the fluorochromes currently used in 2-DGE have been utilized in time-resolved fluorescence applications in these related fields (e.g. CyDyes™ available from GE Healthcare, Uppsala, Sweden, and Alexa-dyes as well as ruthenium chelates such as SYPRO Ruby™, both available from Molecular Probes, Eugene, Oreg., USA (7, 8)). However, the lack of this feature in modern 2-DGE image acquisition equipment is currently prohibiting the use and development of TRF in 2-DGE.
3. Description of the Related Art
Most of the prior art in the field concerns fluorescence resonance energy transfer (FRET) techniques for the study of inter-molecule interactions, molecular stability, or intra-molecular conformational changes, and some concern the use of fluorescent lifetime imaging (FLIM). These include monitoring of polymerized chain reaction (PCR) products (Rintamaki S. et al, Journal of microbiological methods, (2002 August) Vol. 50, No. 3, pp. 313-8) and others for base calling in DNA sequencing (Lassiter S J et al, Analytical chemistry, 2000 Nov. 1, Vol. 72, No. 21, pp. 5373-82). These authors have modified the microscope head in an automated DNA sequencer to allow near-infrared time resolved fluorescent lifetime measurements. Accordingly, the design, capabilities, and utilization of this instrument were of an entirely different character than the invention herein. The instrument modifications performed by this group were designed for classification purposes in order to improve the accuracy and speed of DNA sequencing. In essence, lifetime imaging was utilized to distinguish between two fluorochromes with different lifetimes, representing the presence of different DNA fragments that were fractionated through slab gel electrophoresis. In follow-up studies, the authors expanded the sequencing technology to a polymer microchip platform with a similar purpose (Llopis S D et al, Electrophoresis, 2004 November, Vol. 25, No. 21-22, pp. 3810-9) as well as for reading fluorescent signatures from DNA microarrays (Stryjewski et al Proceedings of SPIE-The International Society for Optical Engineering (2002), 4626 (Biomedical Nanotechnology Architectures and Applications), 201-209). The use of multidimensional time resolved fluorescence for the background subtraction of the invention herein was never used in any of these applications. In contrast, the authors either went to great lengths to investigate which polymer support matrix gave rise to the least amount of background fluorescence in order to maintain a mono-exponential work flow, or alternatively used conventional background correction methods such as time gating or subtraction of the intensity of negative control spots. As such, the use of multi-exponential time resolved fluorescence for specific background subtraction in in-gel (protein) measurements of this invention represents a surprising effect and a novel technology.
An objective of the invention is to provide a new platform for in-gel detection and quantification of fluorescently labeled proteins in global proteome studies. The invention involves the utilization of fluorescent lifetime imaging (FLIM). Other objects and advantages will be more fully apparent from the following disclosure and appended claims.